The primary aim of this project is to identify the Ca2 ion ionophore from sarcoplasmic reticulum Ca2 ion plus Mg2 ion-ATPase and determine its relation to the functional and structural components of the ATPase. Our approach to this problem has been to physically separate the ionophoric site from the ATP splitting site. From these studies we hope to determine how the site which actively transports Ca2 ion is associated with the energy release site of the enzyme. The Mr equals 102,000 Ca2 ion plus Mg2 ion-ATPase has been tryptically digested into four fragments. The first cleavage results in splitting of the enzyme into Mr equals 45,000 and 55,000 fragments. A second cleavage splits the Mr equals 55,000 fragment into Mr equals 20,000 and 30,000 fragments. The four fragments have been purified by gel filtration in sodium dodecylsulfate (SDS) and preparative SDS-polyacrylamide gel electrophoresis. Labelling with (gamma-32P)ATP and N-ethylmaleimide indicate the site of ATP hydrolysis resides in the Mr equals 55,000 and subsequently Mr equals 30,000 fragments. Antibody studies and freeze fracture electron microscopy indicate the Mr equals 55,000 fragment is exposed at the surface of the membrane while the Mr equals 45,000 fragment lies buried in the membrane.